U.S. patent application number 10/228811 was filed with the patent office on 2003-03-20 for design principle for the construction of expression constructs for gene therapy.
This patent application is currently assigned to SOFT GENE GMBH. Invention is credited to Junghans, Claas, Wittig, Burghardt.
Application Number | 20030054392 10/228811 |
Document ID | / |
Family ID | 7812619 |
Filed Date | 2003-03-20 |
United States Patent
Application |
20030054392 |
Kind Code |
A1 |
Wittig, Burghardt ; et
al. |
March 20, 2003 |
Design principle for the construction of expression constructs for
gene therapy
Abstract
The invention concerns an expressible nucleic acid construct,
which contains only the sequence information necessary for
expressing a gene for RNA or protein synthesis. Expression
constructs of this type can be used in gene therapy and genetic
vaccination and avoid many of the risks associated with constructs
today. The invention further concerns the possibility of improving
the conveying of the construct into cells or tissue by covalent
linkage of the construct, for example to particles of peptides.
Inventors: |
Wittig, Burghardt; (Berlin,
DE) ; Junghans, Claas; (Berlin, DE) |
Correspondence
Address: |
NILS H. LJUNGMAN
NILS H. LJUNGMAN & ASSOCIATES
P.O. BOX 130
GREENSBURG
PA
15601-0130
US
|
Assignee: |
SOFT GENE GMBH
|
Family ID: |
7812619 |
Appl. No.: |
10/228811 |
Filed: |
August 27, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10228811 |
Aug 27, 2002 |
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09310842 |
May 12, 1999 |
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6451593 |
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Current U.S.
Class: |
435/6.16 ;
536/23.1 |
Current CPC
Class: |
A61K 48/00 20130101;
Y10S 977/916 20130101; C12N 15/87 20130101 |
Class at
Publication: |
435/6 ;
536/23.1 |
International
Class: |
C12Q 001/68; C07H
021/04 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 13, 1996 |
DE |
196 48 625.4 |
Claims
1. Deoxyribonucleic acid construct for transcription of
RNA-molecules, characterized by a circular strand of
desoxyribonucleic acid comprising a partly complementary,
antiparallel base sequence, so that a dumbbell-shaped construct is
formed, in which the complementary, antiparallel base sequence in
the essential comprises a promotor sequence, a coding sequence and
a polyadenylation signal or another RNA-stabilizing signal, and the
non-complementary sequence comprises two loops of single-stranded
deoxyribonucleic acid, which covalently join the 5'- and 3'-ends of
the complementary, antiparallel strands.
2. Deoxyribonucleic acid construct according to claim 1,
characterized by that said loops consisting of three to seven
nucleotides, and in which one or several of said nucleotides are
covalently modified by carboxylic acid-, amine-, thiole- or
aldehyde functionalities.
3. Deoxyribonucleic acid construct according to claim 2,
characterized by that said chemically modified nucleotides is being
linked to a peptide leading to the directed transport of the
construct into the nucleus.
4. Deoxyribonucleic acid construct according to claim 2,
characterized by that said chemically modified nucleotides is being
linked to a peptide enabling liberation of the construct from the
endosome.
5. Deoxyribonucleic acid construct according to claim 1,
characterized by using a 7SK promoter as said promoter.
6. Deoxyribonucleic acid construct according to claim 1,
characterized by using a CMV promoter as said promoter
7. Deoxyribonucleic acid construct according to at least one of the
claims a to 6, coding for interleukine-7.
8. Deoxyribonucleic acid construct according to at least one of the
claims 1 to 6, coding for interleukine-12 or one or several of its
constituting sub-units.
9. Deoxyribonucleic acid construct according to at least one of the
claims 1 to 6, coding for gm-csf
10. Deoxyribonucleic acid construct according to at least one of
the claims 1 to 6, coding for p16 or p53 protein or fragments
thereof.
11. Desoxyribonucleic acid construct according to at least one of
the claims 1 to 6, coding for peptide fragments of mutated ki-ras,
mutated p53 or bcrabl translocation product with a length of
between 10 and 100 aminoacids.
12. Microprojectile for ballistic transfer of deoxyribonucleic acid
constructs into cells according to claims 1 to 11, in which the
substance to be transported is linked by adsorption or covalent or
ionic binding in such a way to said microprojectile that the
substance to be transported upon passage of the microparticle
through connective tissue, the extracellular liquid or cell layers
is not or not completely desorbed, but remains bound to said
microprojectile until the substance to be transported rests along
with said microprojectile in the target cell.
13. Microprojectile according to claim 12, characterized by that
its material being gold, oxide ceramic, glass ceramic or glass and
said nucleic acid to be transported is bound covalently by thiole-
or disulfide moyeties, ester-, amide-, aldimine-, ketale- or
acetale- or ether functionalities to said microprojectile.
14. Microprojectile according to claim 12 or 13, characterized by
that said microprojectile being made out of an electrically
conducive material and said nucleic acid being linked to said
microprojectile by electrochemically coupling of disulfide or
thiole moieties, employing the microprojectile as electrode.
15. Microprojectile according to claim 12 to 14, characterized by
that said microprojectile being of the size of 0,3 .mu.m to 3
.mu.m.
16. Use of a nucleic acid construct according to one or more of the
claims 1 to 11 in ex-vivo gene therapy.
17. Use of a microprojectile according to one or more of the claims
12 to 15 in ex-vivo gene therapy.
18. Use of said deoxyribonucleic acid constructs according to one
or several of the claims 1 to 6 as vaccine for prevention of
infectious disease in humans or animals.
Description
DESCRIPTION
[0001] The invention concerns a design principle for a minimalistic
expression construct which contains no genetic information other
than the information to be expressed apart from promotor and
terminator sequences which are necessary for the control of
expression Such minimal expression constructs are to be used for
molecular-medical applications, specifically genetic vaccination,
tumor therapy, and -prophylaxis.
[0002] The design principle is to be used for the construction of
expression constructs for the expression of MHC-I or MHC-II
presentable peptides, cytokines, or components of the regulation of
the cell cycle, or for the synthesis of regulative RNA molecules
and antisense RNA, ribozyme or mRNA-editing-RNA. Furthermore, an
important aspect of the invention is that the construction
principle allows for the covalent linking of the expression
construct, e.g. with peptides, proteins, carbohydrates or
glycopeptide ligands, as well as particles which allow for the
transfer of the constructs into cells by ballistic transfer
especially into dermis, muscle tissue, pancreas, and the liver.
[0003] The invention is to be used especially in two related
fields: somatic gene therapy and genetic vaccination. These two
meet in the field of immuno gene therapy of oncological conditions.
Whereas classical gene therapy intends to substitute missing or
defective genes, immuno gene therapy intends to activate the immune
system of the patient against tumor specific antigens. In malignant
melanoma and some other tumors, a number of tumorspecific antigens
have been identified which can be recognized by cytotoxic
T-lymphocytes (Van den Eynde B. and Brichard V. G., Current Opinion
in Immunology (1995) 7: 674-681). In most cases these are fragments
of mutated proteins, which are either relevant for tumor
development and -promotion, or are fragments of proteins from a
changed metabolism of the tumor cell (Stuber et al., Eur. J.
Immunol. (1994) 24: 765-768). In the case of melanoma, the
presented peptides often derive from proteins from the
melanocyte-specific differentiation. Approaches which make use of
the activation of the immune system against such tumor specific
antigens are in need of methods which enable the antigenic epitopes
to be overexpressed in non-tumor cells, such as antigen-presenting
cells (macrophages, dendritic cells) Alternatively, genes which
control the expression of peptide-presenting proteins, such as
CIITA or ICSBP are of great importance.
[0004] Laboratory experiments and clinical studies, in which such
peptides have been used for the induction or amplification of a
tumor specific cytotoxic response, concentrate on conventional
vaccination protocols, in which the corresponding peptides are
being used (Strominger J., Nature Medicine, (1995) 1:1.179-1.183).
Alternatively, antigen-presenting cells such as dendritic cells,
were incubated with high concentrations of such peptides. Thereby,
the peptides originally present on the MHC-complex were exchanged
for tumor specific peptides (Grabbe et al., Immunology Today (1995)
16:117-121).
[0005] The term genetic vaccination (immunization) describes the
utilization of an experimental finding which first was debated as a
scientific artefact, but has recently been corroborated in a number
of biomedical problems (Piatak et al., Science 259 (1993):
1745-1749). If an expression plasmid for mammalian cells is
injected into skin or muscle, there is, albeit in very low
efficiency, an expression of the corresponding gene close to the
injection site. If the expression product is a protein alien to the
organism (xenogenic or allogenic protein), uptake and presentation
of fragments of the expressed protein (oligopeptide) by
antigen-presenting cells (APC) takes place, probably by way of
local inflammation, Depending on local cytokine patterns and the
type of cells in which the plasmid is expressed (presentation by
MHC-I or MHC-II), there is an induction of an immune reaction along
the T.sub.H1 or T.sub.H2 pathway (Wang et al., Human Gene Therapy 6
(1995): 407-418), which eventually leads to the proliferation of
cytotoxic T-cells or to the production of soluble antibodies. The
transfection of dendritic cells with expression constructs for
antigenic peptides ex-vivo is included in the term genetic
vaccination in this context (Schadendorf et al. Molecular Medicine
Today, 2 (1996): 144-145).
[0006] Such genetic vaccination avoids the numerous risks of
conventional immunization approaches. Many approaches are known in
gene therapy that are designed to effect therapeutic or
prophylactic effects by the transfer of genetic information into
cells. These approaches have not only been demonstrated in animal
experiments, but also in numerous clinical studies in patients, an
example being the so-called ballisto magnetic vector system
(EP0686697 A2) for the transfection of conventional, plasmid-based
expression constructs. The ballisto magnetic vector system was
employed by the inventors of this application in three clinical
phase I/II studies for the production of interleukin-7 (IL-7),
interleukin-12 (IL-12) or granulocyte-macrophage-colony stimulating
factor (GM-CSF) expressing tumor cells. In the case of expression
of IL-12, separate expression constructs for the genes of the two
IL-12 subunits were transferred ballisto-magnetically at the same
time.
[0007] With the maturing of this new discipline, however, the
methodological repertoire for gene therapy demands critical
inspection. A fundamental aspect of this question is the sequence
information contained in conventionally employed DNA constructs .
If such expression constructs are to be employed in a great number
of patients, and possibly more than once, safety aspects,
especially those related to immunological concerns, will come to
bear heavily. The conventionally used expression constructs are
derivatives of eucaryotic expression plasmids. These have two
fundamental disadvantages: their size, which inhibits fast
transport into the cell's nucleus, and the presence of sequences
which are not needed for the intended use. Expression constructs
used so far contain constitutively expressed genes, i.e. for
resistance against cytostatica which serve as selection markers,
and in some cases sequences for the episomal replication in the
target cell. The expression of these genes leads to an unwanted
background of transfected genetic information. Furthermore, apart
from the promotor-gene-terminator structure which is to be
expressed, these constructs carry at least the sequences needed for
bacterial replication, since the plasmids are propagated in
bacteria. These sequences are not needed for the intended use,
either.
[0008] It is obvious that conventional expression constructs lead
not only to the expression of the desired gene, but also to the
biosynthesis of xenogenic proteins, even if their prokaryotic
promoters show very low activity in mammalian cells. With longer or
repeated application it can be assumed that the desired immune
response is masked by such contaminating gene products, and
significant immunological complications can occur.
[0009] Another problem in the application of gene therapeutic
methods concerns the method by which the genetic material to be
transferred is brought into the cell. For reasons of efficiency,
immunological safety, and broad applicability across a wide
spectrum of cell types, the method of ballistic transfer is
preferred. A fundamental advantage of ballistic transfer, compared
to alternative transfection methods, is that the method is
applicable across a broad spectrum of different cells or tissues.
Another disadvantage of methods currently used for the transfection
of eukaryotic cells, such as electroporation or lipofaction, is
that the treatment brings the substance to be transported only
across the plasma membrane, the first barrier, which shields the
cell from its environment. However, for most substances interacting
with the regulative function of the cell, it is important to get
from the cytoplasma across the nuclear membrane into the nucleus.
This membrane is biophysically fundamentally different from the
plasma membrane, and methods such as electroporation or lipofection
do not lead to a passage through this membrane. The reason why
these methods nonetheless lead to expression of recombinant nucleic
acid constructs transfected into the cells in a part of the cell
population, is the fact that in the act of cell division, the
nuclear membrane is rendered permeable In consequence, methods such
as electroporation or lipofaction only lead to transfection of
cells which divide. Therefore these methods are not applicable to
the transfection of many slowly or non-dividing cells, which can be
interesting in the context of gene therapy, such as stem cells of
the immune system or the heamatopoietic system, muscle cells, cells
of exocrine or endocrine organs and their accompanying cells. The
also commonly used and very efficient transfection method of
retroviral transport of genetic material suffers the great
disadvantage of targeting the tranfected cells for a possible
cytotoxic reaction by the host organism, which probably limits the
applicability of this method for gene therapeutic approaches.
[0010] The method of ballistic transfer has been used for the
ex-vivo treatment of autologous and allogenic patient cells (Mahvi
et.al.; Human Gene Therapy 7 (1996) 1535-43). However, when
treating cells in tissue, a method which should be advantageous
especially for the oncotherapeutic treatment of solid tumors or the
mass prophylaxis against infections by genetic vaccination, the
state of the art has disadvantages. The method of ballistic
transfer makes use of DNA adsorbed to microprojectiles. When
transfecting skin or other tissues, the penetration depth of the
DNA constructs is lower than the penetration depth of the
projectiles. DNA is desorbed soon after impact on the tissue. Only
the uppermost tissue layer in the direction of the projectiles is
transfected, although the projectiles themselves enter much deeper
into the tissue. When transfecting solid tumor tissue (colon
carcinoma, rectum carcinoma, reno-cell carcinoma and others), it
has been found that, with suitable adaption of the parameters, the
microprojectiles enter up to five cell layers deep into tissue
slices. The transfected cells, however, (visible as fluorescent
cells when transfected with a recombinant expression construct
containing a green fluorescent protein from aequrea sectoria) were
all found in the uppermost cell layer facing the impact of the
microprojectiles. A more stable coupling of the DNA constructs to
the surface of the microprojectiles would be desirable in order to
avoid the desorption of the substance to be transported. In this
way only, the application of gene therapeutic approaches to solid
tumors would be realistic, since only the transfection of tumor
slices in the depth of the tissue enables a sufficient number of
treated cells to be achieved. It is also imaginable that a
combination of different coupling protocols enables the release of
different genetic information within the same cell population at
different timepoints. For these and numerous other applications,
microparticles which bring the substance to be transported all the
way into the hit cell and then make the substance available to the
cell, would be very desirable.
[0011] U.S. Pat. No. 5,584,807 (McCabe) describes an instrument in
form of a gas pressure operated gun for the introduction of genetic
material into biological tissue, in which gold particles are used
as carrier material for the genetic information, without making
reference to the nature of the genetic material in particular. U.S.
Pat. Nos. 5,580,859 and 5,589,466 (Felgner) describe a method for
the introduction of DNA into mammalian cells in the context of gene
therapy. Naked DNA sequences coding for physiologically active
proteins, peptides or polypeptides and are under the control of a
promotor are injected directly into cells. Naked DNA refers to
sequences that are free of other genetic material like viral
sequences. DNA is expressed in these cells and serves as
vaccine.
[0012] WO 96/26270 (Rhne-Poulenc Rorer S. A.) describes a circular
double-stranded (supercoiled) DNA molecule, containing an
expression cassette coding for a gene and controlled by a promoter
and a terminator. This system is employed in vaccination in the
context of gene therapy, also.
[0013] EP 0 686 697 A2 (Soft Gene) concerns a method for the
enrichment of cells modified by ballistic transfer, and describes
the technological background, the related problems, and the
solutions found so far. The basic method of ballistic transfer is
described herein. A device useful for the execution of this method
is described in EP 0 732 395 A1.
[0014] The ballistic particles are gold particles with a diameter
of either 1 .mu.m or 1,5 .mu.m (EP 0 686 697 A2), chosen depending
upon the cell type. These gold particles are coated with
superparamagnetic particles of roughly 30 nm diameter. The
superparamagnetic particles at the same time furnish a useful
surface for the coating with biomolecules. The use of magnetic
particles enables subseqent separation.
[0015] Furthermore, dumbbell-shaped nucleic acid constructs are
known that are characterized by the following features: They are
short (10-50 bp double-stranded DNA) nucleic acid constructs, which
were made for structure research or as double-stranded oligomers
with improved nuclease resitence used for scavenging of sequence
specific DNA ligands (Clusel et. al.; Nucleic Acids Res. 21 (1993):
3405-11; Lim et. al., Nucleic Acids Res. 25 (1997): 575-81).
[0016] Longer DNA molecules, which can exist throughout parts of
their replication cycles as dumbbells, are known in nature as
mitochondrial genomes of some species, such as ciliatae and yeasts
(Dinuel et. al., Molecular and Cell Biology, 13 (1993): 2315-23).
These molecules are about 50 kb in size and have a very complex
genetical structure. Likewise, a closed covalent linear structure
is known from vaccinia virus.
[0017] Peptide-nucleic acid-linkages with localization sequences
are known for short DNA oligomers. Morris et al. (Nucleic Acids
Res. 25 (1997): 2730-36) describe the coupling of oligomers 18-36
base pairs in length, with a 27 amino acid residues containing
peptide, which contains the nuclear localization sequence from SV40
as well as a signal peptide from HIV-gp41 responsible for the
fusion with CD4-positive cells.
[0018] The use of peptide chains for crossing the endosomal
membrane has been investigated by several groups. The 23 N-terminal
amino acids of haemagglutinine were adsorbed by non-covalent
interactions to expression plasmids in order to facilitate the
uptake of these complexes into the cytosol after endosomal uptake
(Plank et.al., J.Biol.Chem. 269, 12918, (1994)). The covalent
attachment of antisense desoxyoligonucleotides to haemagglutinine
peptide is described by Bongartz et al. (Nuc.Acids Res. 22, 4681,
1994).
[0019] Based on this state of the art, it is the objective of the
invention presented here to develop an expression construct that
contains only the information necessary to be expressed, and to
provide means for the transport into a cell, which is to be treated
therapeutically.
[0020] This objective is reached using the features of claims 1 and
13. According to the invention, double-stranded DNA-expression
constructs, which are to be transported, are modified in such
fashion that both anti-parallel strands of the DNA-polymer,
containing the coding sequence and the promotor and terminator
sequences necessary for its expression, are linked by loops of
single stranded desoxyribonucleotides at both ends in such a way,
that a continuous covalently closed molecule results. Preferably,
said loop contains 3 to 7 nucleotides. In FIG. 1, such a construct
is shown schematically. Such expression constructs are employed for
the expression of MHC-I or MHC-II presentable peptides, cytokines,
or components of the regulation of the cell cycle, or for the
synthesis of regulative RNA-molecules, such as antisense-RNA,
ribozymes or mRNA-editing RNA. Since the nucleic acid is covalently
closed on both ends and no free hydroxyl-groups are available for
nucleolytic cleavage, the molecule has a much higher stability
against intra- and inter-cellular nucleases, and thus a longer
halftime in the body or the cell. This advantage is especially
important in the application in-vivo.
[0021] Furthermore, according to the invention, said loop linking
the strands can contain one or more modified bases, said bases
containing chemical functions, which allow the coupling of the
molecule with a solid base, preferably amino-, carboxylic acid-,
thiol-, or disulfide-modifications. Said modifications are
covalently linked by known synthetic steps with corresponding
carboxylic acid-, aldehyde-, amine-, thiole-, or other functions,
or directly with a gold surface of a microprojectile for ballistic
transfer. It can be imagined that a combination of different
linking methods facilitates the release of a plurality of genetic
information within the same cell population at different
timepoints.
[0022] Apart from the aspect of easier chemical linking to the
surface of the microprojectile, said nucleic acid construct
presents another advantage: nucleic acid constructs currently used
in transfection in gene therapy, are produced in bacteria and
carry, besides the sequences relevant in context of their
therapeutical use, other sequences, which are only needed for the
amplification of the nucleic acid constructs in bacteria. These
sequences are an unknown risk for the patient who is to be treated,
as it is not known whether and how these affect the organism. Such
sequences, which not solely serve the primary objective of
transfection in the target cells, can be excised from nucleic acids
amplified in bacteria by restriction endonuclease digestion prior
to transfection, and can be substituted by covalent linkage of
short ends of desoxyribonucleic acid, said ends possibly being
modified. According to another aspect of the invention, DNA to be
transported into the cell can be obtained by polymerase chain
reaction with chemically modified primers, so that the products of
the polymerase chain reaction contain the chemical modifications
needed for binding to the micro-projectile. An advantage of the
construction principle according to the invention over current
expression vectors is, that the resulting constructs contain only
the sequence needed for the expression of the target gene.
[0023] Another aspect of the invention is that a loop of single
stranded desoxyribonucleotides on either end of the molecules
allows for the introduction of chemical modifications in such a way
that non-nucleic acid ligands can be covalently linked to the
nucleic acid expression construct. This way for example, peptides
needed for the nuclear localization of the expression constructs
can be linked to the construct in such a way, that after entering
the cytosol of a cell said construct is transported by the
translocation apparatus of the cell into nuclear compartments where
it can be transcribed. Thereby, the constraints mentioned above
concerning some transfection methods would be dealt with. Likewise
the direct linking of the construct to peptides-, glycopeptide-, or
carbohydrate-ligands which facilitate the entrance of the construct
by cell-specific surface receptors is facilitated.
[0024] Specifically, according to the invention, double-stranded
anti-parallel DNA expression constructs are modified in such a way
that the ends of the double strands each contain a disulfid bridge
linking the strands on both ends over covalently bonded alkyl
groups bound to the 5'-end of one strand and the 3'-end of the
other strand.
[0025] Any eucariotic promoter sequence can be employed for the
control of transcription of the expression plasmid. Especially
advantageous are short promoters with a high transcription rate
transcribable in a multitude of cells, e.g. the "immediate early
promotor" from cytomegalo virus (CMV) promoter RNA-polymerase
III-depending promoters such as the 7SK-promoter or the U6-promoter
are of advantage for the transcription of genes coding for RNA.
Such promoter sequences can result in the expression of short
antisense-RNAs, ribozymes, and artificial mRNA in vivo.
RNA-polymerase III produces significantly more copies of RNA than
polymerase II and has an exact termination signal, a feature of
special advantage. Said promotor sequences are characterized by
their shortness, leading to small corresponding dumbbell expression
constructs, a feature favourable to the entry of said expression
constructs into the cell's nucleus.
[0026] The invention also concerns the transport of nucleic acids
into cells. According to the invention, nucleic acids are
transferred into cells by linking the nucleic acids to the surface
of the micro-projectile carrying the nucleic acids into cells by
adsorption, covalent or ionic interaction, in such a way that said
nucleic acids are not or not completely desorbed upon passage of
the microprojectile through connected tissue into tissue fluid or
cell layers, but remain linked to the microprojectiles until said
microprojectiles come to rest with said nucleic acids in the target
cells. This has the advantage that the substance is provided to a
cell either in its bound state, or is desorbed in a process slow in
comparison to the time of entry and is provided to the cell in a
desorbed state, depending on the mode of binding.
[0027] The binding of thioles or disulfides to gold surfaces is
well researched and described (G. M. Whitesides et al., Langmuir 10
(1994): 1825-1831). The nucleic acids to be transported are
adsorbed preferably to micro-projectiles made of gold, however, by
providing the nucleic acids with thiole or disulfide groups by
covalent modification with publicly available reagents, and then
adsorbed through their thiole or disulfide groups, or covalently
bound to the micro-projectile by anodic oxidation of the thiole or
disulfide functions employing the gold of the micro-projectile as
anode. According to the invention, the substance to be transported
is bound by sulfur-gold linkage or by disulfide linkage to the
micro-projectile. When employing micro-projectiles made of gold,
the substance to be transported is modified by molecules containing
thiole groups or disulfide bridges, if it does not already contain
thiole or disulfide groups able to bind by themself. Said substance
is then bound to the gold surface of the micro-projectile by
chemisorption. The resulting gold-sulfur linkage is sufficiently
strong to carry the molecules to be transported through several
cell layers.
[0028] Since the cell contains molecules comprising thiole groups,
above all the ubiquitous glutathion, an equilibrium reaction of the
thiole groups on the gold surface leads to the slow desorption of
the chemisorbed molecule from the surface of the micro-projectile.
Thereby, the transported substance is freely available to the cell
after desorption. Furthermore, according to the invention, when
employing micro-projectiles made of gold, the molecule to be
transported containing thiole or disulfide bridges can be bound
covalently to the micro-projectile by anodic oxidation of the
thiole or disulfide functions employing the gold micro-projectile
as anode
[0029] According to the invention, when employing micro-projectiles
made of oxidic ceramics, glass ceramic, or glass, the molecule to
be transported is bound by ester, amide, aldimine, ketal, acetal or
ether linkage, or other functionalities known to the organic
chemist for binding of molecules to a solid surface. The numerous
silane reagents used for the modification of silicon oxyde phases
can be employed here.
[0030] The invention is used for ex-vivo gene therapy. Preferably,
interleukin-7 (IL-7) and interleukin-12 (IL-12) proteins and their
subunits are expressed, as are interleukines,
granulocyte-makrophage-colo- ny-stimulating factor (GM-CSF), cell
surface antigens and ligands of immune controlling or -modifying
lymphocyte antigens like CD40, B7-1, and B7-2, proteins of the
MHC-complexes I or II or .beta.-2 microglobulin, interferone
consensus sequence binding protein ICSBP, CIITA, Flt3, or entire
proteins or fragments thereof of presentable epitopes from tumor
specific expressed mutated or non-mutated proteins, e.g.
Ki-RAS-fragments, p16 and p53, or bcr-abl product. The use of micro
projectiles which are ail linked with constructs of the same type
is preferred, but cocktails (mixtures) of micro-projectiles which
are each linked to different constructs are possible, as are
micro-projectiles which are each linked with a cocktail of
different constructs.
[0031] The desoxyribonucleic acid construct according to the
invention is preferably employed as vaccine for the treatment of
infectious diseases in humans and animals, e.g. malaria and
influenza.
[0032] More advantageous features are contained in the subclaims.
The invention is depicted in the attached figures and is described
more closely in the following examples.
[0033] FIG. 1 shows a schematic outline of the construction
concept, whereas FIG. 1.1. shows the confirmation of the covalently
closed phosphate-sugar-backbone; the number of base pairs depicted
does not necessarily show the length of the constructs but only
serves as example of the principle; FIG. 1.2. shows the functional
structure of one of several possible expression constructs;
[0034] FIG. 2 shows the synthesis of the constructs
schematically.
EXAMPLE 1
Synthesis of the Expression Constructs
[0035] The fundamental construction principle is depicted
schematically in FIG. 2 and is as follows:
[0036] 1.1 Synthesis from Vector
[0037] The gene to be expressed, e.g. granulocyte-macrophage
stimulating factor (GM-CSF), is amplified from cDNA using suitable
primers by PCR (FIG. 2(1)) and recombined into a suitable plasmid
vector (FIG. 2(2)). After sequencing and confirmation of the target
sequence, the sequence to be expressed is amplified from said
plasmid vector by means of two primer sequences
(oligodesoxynucleotides carrying on the 5'-end of their sequence
restriction enzyme recognition sites) (FIG. 2(3)). The resulting
amplification product is digested with said endonucleases, for
which a recognition site was provided on said primers. After
isolation of the amplified fragment from an agarose-gel, said
fragment is recombined into an expression plasmid, which is
amplifiable in bacteria, and in which the gene to be expressed is
located in the desired orientation in the context of the sequences
controlling expression contained in said expression plasmid (FIG.
2(4)).
[0038] Said expression plasmid is amplified in bacteria and
isolated according to methods known in the art. After digestion
with restriction endonucleases, the recognition sites of which are
flanking the sequence which is to be contained in the
dumbbell-shaped expression constructs, the restriction fragments
are separately isolated by methods of anion exchange chromatography
(FIG. 2(5)), and are subsequently ligated to hairpin-forming
self-hybridizing oligodesoxynucleotides (short DNA-molecules
obtained by automated chemical DNA synthesis, which can form
stem-loop-structures based on their self-complimentarity; these
molecules will later form the covalently closed ends of the
dumbbell-shaped DNA-molecules), which contain an single-stranded
overlap compatible with the overlap of the construct obtained by
the digestion with endonucleases (FIG. 2(6)). After separation of
excess hairpin desoxynucleotides by anion exchange chromatography,
the constructs according to the invention are obtained.
[0039] 1.2. Synthesis from PCR-product
[0040] Alternatively, the construct is amplified directly by
polymerase chain reaction, the primer oligodesoxynucleotides
carrying recognition sites for restriction endonucleases on the
5-prime ends of their sequence (FIG. 2(7)). After separation of the
primers by anion exchange chromatography, the resulting amplificate
is digested with said endonucleases, for which a recognition
sequence was provided on said primer oligodesoxynucleotides. After
separation of the smaller restriction fragments, the construct is
ligated to short hairpin-formed self-hybridizing
oligodesoxynucleotides, said hairpin-formed desoxynucleotides
providing a overhang able to hybridize to the overhang resulting
from the restriction enzyme digestion of the construct (FIG. 2(8)).
After separation of excess hairpin-desoxyoligonucleotides by anion
exchange chromatography, the constructs according to the invention
are obtained.
[0041] An expression construct consisting of the sequence for
gm-csf under control of the "early immediate promotor" from CMV and
the polyadenylation sequence from SV40, was obtained from plasmid
mtv-GM-CSF by complete digestion with EcoRI and HindIII. The
smaller fragment (1290 bp) was isolated by anion exchange
chromatography (stationary phase Merck fractogel EMD-DMAE; 25 mM
Tris/HCl pH8; 0-1 M NaCl), and after concentration and desalting,
was ligated with a 200-fold molar excess of 5' phosphorylated
hairpin-desoxyribonucleotides AATTCGGCCGGCCGTTTTCGGCCGG- CCG and
AGCTTGGCCGGCCGTTTTCGGCCGGCCA (TIB Molbiol, Berlin) in the presence
of 25 U/ml T4-DNA-ligase overnight at room temperature. The
reaction was stopped by heating to 60.degree. C. The construct
ligated to the desoxyoligoribonucleotide was separated from excess
desoxyoligoribonucleotide by anion exchange chromatography,
concentrated by ethanol precipitation, dissolved in water and
applied to sterile primary colon carcinoma cells using the
ballistic transfer according to a published method.
EXAMPLE 2
in vivo Expression
[0042] Ballistic Transfer of GM-CSF into K562
[0043] 30 .mu.l of a suspension of gold particles (1.6 .mu.m
diameter, supplied by Bio-Rad, Hercules, Calif., USA, concentration
of the suspension: 30 mg/ml) are transferred to a macro
carrier-polymer sheet (Bio-Rad). The gold is allowed to sediment,
and the supernatant is cautiously removed. Onto the wetted surface,
30 .mu.l of a 1+3 mixture of a suspension of colloidal magnetic
particles (mean diameter 65 nm; Miltenyi GmbH, Bergisch-Gladbach,
Germany; used as supplied; concentration unknown) and
GM-CSF-expression-dumbbell-construct (example 1) are pipetted. The
sedimented gold is re-suspended in said mixture and allowed to
re-sediment. The supernatant liquid is removed and the gold
particles are allowed to dry. 300 .mu.l polylysine are transferred
to the center of a petri-dish (3,5 cm), allowed to rest for 30 min
and washed off with PBS-medium. 100.000-200.000 cells
(erytroleukemia cell line K562) are transferred onto the
polylysine-coated surface of the petri-dish in 300 .mu.l
RPMI-medium (10% FCS), and allowed to rest for 10 min. 2 ml
RPMI-medium (10% FCS) are added, and the cells are incubated 1-2 h
in an incubator.
[0044] Ballistic transfer is conducted according to the
manufacturers with a Biolystic PDS 1000/C (Bio-Rad, Hercules,
Calif., USA). The rupture disk employed corresponds to a pressure
of 1100 psi. The pressure of the vacuum cell is 508 mm Hg. Magnetic
separation is conducted as published (EP 0732 395A1); control of
successful transfection is performed by GM-CSF-ELISA.
EXAMPLE 3
Example for Synthesis of a Expression-construct with Nuclear
Localization Sequence
[0045] An expression construct consisting of the gene for a green
fluorescent protein under control of the early immediate promoter
from CMV and the polyadenylation sequence of SV40 (pEGFP, Clontech
Inc.) was obtained by restricition enzyme digestion with EcoRI and
HindIII. The smaller fragment was isolated by anion exchange
chromatography (stat phase: Merck Fractogel EMD-DMAE; 25 mM
Tris/HCl pH 8; 0-1M NaCl) and ligated after concentration and
desalting with a 200-fold molar excess of 5'-phosphorylated
hairpin-desoxyoligoribonucleotides AATTCGGCCGGCCGTXTCGGCCGGCCG and
AGCTTGGCCGGCCGTXTCGGCCGGCCA in the presence of 25 u/ml
T4-DNA-Ligase overnight at room temperature (X signifies the
peptide modification: Amino Uracil coupled to the peptide by amide
function (TIB-Molbiol, Berlin)) The reaction was stopped by heating
to 60.degree. C. The construct ligated to the
amino-desoxy-uracil-modified desoxyoligonucleotide was separated
from excess hairpin desoxynucleotides by anion exchange
chromatography, concentrated by ethanol precipitation and dissolved
in water. 1 .mu.g of the thiol-modified construct was incubated
with 1 mg micro-projectiles (spherical gold, mean diameter 1 .mu.m,
Bio-Rad, Hercules, Calif.) in water over night at room temperature.
The gold particles were washed twice with water and applied to
adherent ceratinocytes with the ballistic transfer according to the
known procedure.
EXAMPLE 4
Synthesis of Nucleic-acid-modified Gold Particles
[0046] An expression construct, which consists of the sequence for
gm-csf under control of the "early immediate promoter" from CMV and
the polyadenylation sequence from SV40 was excised from the plasmid
mtv-gmcsf by complete digest with EcoRI and HindIII. The smaller
fragment (1290 bp) was isolated by anion exchange chromatography
(stat. phase : Merck Fractogel EMD-DMAE; 25 mM Tris/HCl pH 8; 0-1 M
NaCl) and following concentration and desalting ligated to a
200-fold molar excess of 5' phosphorylated hairpin
desoxyoligoribonucleotides AATTCGGCCGGCCGTXTCGGCCG- GCCG and
AGCTTGGCCGGCCGTXTCGGCCGGCCA (X specifies the thiol modifier C6 S-S
(TIB-Molbiol, Berlin)) in the presence of 25 u/ml T4 DNA Ligase and
incubated over night at room temperature. The reaction was stopped
by heating to 60.degree. C. The construct ligated to the thiol
desoxyribonucleotide was separated from excess thiol modified
desoxyribonucleotide by anion exchange chromatography, and resolved
in water. 1 .mu.g of the thiol modified construt was incubated over
night with 1 mg microprojectiles (shperical gold, mean diameter 1
.mu.m, Bio-Rad, Hercules, Calif.) in water. The gold particles were
washed twice with water and used for ballistic transfer into
sterile primary coloncarcinoma cells ( see Expl. 5).
EXAMPLE 5
Ballistic Transfer to Solid Tumor Tissue
[0047] Sterile primary colon carcinoma tissue was removed
surgically and cooled on ice. Necrotic parts and connective tissue
is removed as much as possible. Pieces of ca. 1 cm.sup.2 surface
are excised from the tumor, washed in ice-cold PBS and fixated on
the sample holder of a tissue slicer (vibratome 1000 sectioning
system; TPI, St. Louis, Mo.) with tissue glue. The tumor is sliced
into slices of 500 .mu.m thickness. The slices are stored in
ice-cold PBS and transfected as soon as possible. 30 .mu.l of a
suspension of GM-CSF-expression construct-coated gold particles and
colloidal magnetic particles (mean diameter: 65 nm--Miltenyi GmbH,
Bergisch-Gladbach) are pipeted onto a macro carrier polymer sheet
(Bio-Rad). The gold is allowed to sediment, the supernatant removed
and the gold particles are allowed to dry. The procedure of
ballistic transfer is identical with the procedure described in
example 2. Both sides of the tumor slice are transfected. After
transfection, the slice is passed twice through a cell sieve, and
the cells are separated as described.
[0048] Magnetic separation is performed according to the published
protocol (EP 0 732 395 A1); the success of the transfection is
controlled by GM-CSF-ELISA.
EXAMPLE 6
Synthesis of Nucleic Acid-modified Aluminum Particles
[0049] 5 .mu.g of an expression construct consisting of the gene
for a green fluorescent protein under control of the early
immediate promoter from CMV and the polyadenylation sequence of
SV40 (pEGFP, Clontech Inc.) was obtained by restricition enzyme
digestion with EcoRI and HindIII. The smaller fragment was isolated
by anion exchange chromatography (stat.phase: Merck Fracto-gel
EMD-DMAE; 25 mM Tris/HCl pH 8; 0-1M NaCl) and ligated after
concentration and desalting with a 200-fold molar excess of
5'-phosphorylated hair-pin-desoxyoligoribonucleotides
AATTCGGCCGGCCGTYTCGGCCGGCCG and AGCTTGGCCGGCCGTYTCGGCCGGCCA (Y
signifies the carboxylic acid modified thymidinedesoxynucleotide
(TIB-Molbiol, Berlin)) in the presence of 25 U/ml T4-DNA-Ligase
overnight at room temperature
[0050] 1 g aluminiumoxyde particles (mean diameter 1,0 .mu.m) were
refluxed in a solution of tri-etoxaminopropylsilane in toluene (2%)
overnight. The solid matter is filtrated, washed with toluene and
ethanol, dried and ground. 5 mg of the resulting amino-modified
aluminiumoxyde are reacted in 100 ml aqueous carbonate buffer (pH
8,0) with 4 .mu.g of the carbonic-acid-modified construct in the
presence of 50 .mu.M 1-ethyl-3-(3-dimethylaminopropyld)carbodiimide
and 50 mM N-hydroxysuccinimide for 2 h at room temperature. The
resulting nucleic-acid modified microparticles can be transported
to cells by acceleration in a suitable apparatus, as described in
DE 195 10 696 and EP 0 732 395 A1, whereby the information
contained in the transported constructs is made available to the
cells
Sequence CWU 1
1
13 1 1078 DNA Artificial Sequence gene (1)..(1078) bcr3=abl2; Oligo
DNA Dumbbell 1 ttcggccggc caagcttaac cgtattaccg ccatgcatta
gttattaata gtaatcaatt 60 acggggtcat tagttcatag cccatatatg
gagttccgcg ttacataact tacggtaaat 120 ggcccgcctg gctgaccgcc
caacgacccc cgcccattga cgtcaataat gacgtatgtt 180 cccatagtaa
cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa 240
actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc
300 aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg
ggactttcct 360 acttggcagt acatctacgt attagtcatc gctattacca
tggtgatgcg gttttggcag 420 tacatcaatg ggcgtggata gcggtttgac
tcacggggat ttccaagtct ccaccccatt 480 gacgtcaatg ggagtttgtt
ttggcaccaa aatcaacggg actttccaaa atgtcgtaac 540 aactccgccc
cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc 600
agagctggtt tagtgaaccg tcagatggta ccatgctgac caactcgtgt gtgaaactcc
660 agactgtcca cagcattccg ctgaccatca ataaggaaga tgatgagtct
ccggggctct 720 atgggtttct gaatgtcatc gtccactcag ccactggatt
taagcagagt tcaaaagccc 780 ttcagcggcc agtagcatct gactttgagc
ctcagggtct gagttaagag ctcataatca 840 gccataccac atttgtagag
gttttacttg ctttaaaaaa cctcccacac ctccccctga 900 acctgaaaca
taaaatgaat gcaattgttg ttgttaactt gtttattgca gcttataatg 960
gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
1020 ctagttgtgg tttgtccaaa ctcatcaatg tatcttaacg cgaattcggc
cggccgtt 1078 2 1645 DNA Artificial Sequence gene (1)..(1645)
Interleukin-12 (IL-12, p35-subunit); Oligo DNA Dumbbell 2
ttcggccggc caagcttaac cgtattaccg ccatgcatta gttattaata gtaatcaatt
60 acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact
tacggtaaat 120 ggcccgcctg gctgaccgcc caacgacccc cgcccattga
cgtcaataat gacgtatgtt 180 cccatagtaa cgccaatagg gactttccat
tgacgtcaat gggtggagta tttacggtaa 240 actgcccact tggcagtaca
tcaagtgtat catatgccaa gtacgccccc tattgacgtc 300 aatgacggta
aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct 360
acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag
420 tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct
ccaccccatt 480 gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg
actttccaaa atgtcgtaac 540 aactccgccc cattgacgca aatgggcggt
aggcgtgtac ggtgggaggt ctatataagc 600 agagctggtt tagtgaaccg
tcagatggta ccatgtggcc ccctgggtca gcctcccagc 660 caccgccctc
acctgccgcg gccacaggtc tgcatccagc ggctcgccct gtgtccctgc 720
agtgccggct cagcatgtgt ccagcgcgca gcctcctcct tgtcgctacc ctggtcctcc
780 tggaccacct cagtttggcc agaaacctcc ccgtggccac tccagaccca
ggaatgttcc 840 catgccttca ccactcccaa aacctgctga gggccgtcag
caacatgctc cagaaggcca 900 gacaaactct agaattttac ccttgcactt
ctgaagagat tgatcatgaa gatatcacaa 960 aagataaaac cagcacagtg
gaggcctgtt taccattgga attaaccaag aatgagagtt 1020 gcctaaattc
cagagagacc tctttcataa ctaatgggag ttgcctggcc tccagaaaga 1080
cctcttttat gatggccctg tgccttagta gtatttatga agacttgaag atgtaccagg
1140 tggagttcaa gaccatgaat gcaaaacttc tgatggatcc taagaggcag
atctttctag 1200 atcaaaacat gctggcagtt attgatgagc tgatgcaggc
cctgaatttc aacagtgaga 1260 ctgtgccaca aaaatcctcc cttgaagaac
cggattttta taaaactaaa atcaagctct 1320 gcatacttct tcatgctttc
agaattcggg cagtgactat tgatagagtg atgagctatc 1380 tgaatgcttc
ctaagagctc ataatcagcc ataccacatt tgtagaggtt ttacttgctt 1440
taaaaaacct cccacacctc cccctgaacc tgaaacataa aatgaatgca attgttgttg
1500 ttaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc
acaaatttca 1560 caaataaagc atttttttca ctgcattcta gttgtggttt
gtccaaactc atcaatgtat 1620 cttaacgcga attcggccgg ccgtt 1645 3 1318
DNA Artificial Sequence gene (1)..(1318) GM-CSF; Oligo DNA Dumbbell
3 ttcggccggc caagcttaac cgtattaccg ccatgcatta gttattaata gtaatcaatt
60 acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact
tacggtaaat 120 4 1870 DNA Artificial Sequence gene (1)..(1870)
Interleukin-12 (IL-12, p40-subunit); Oligo DNA Dumbbell 4
ttcggccggc caagcttaac cgtattaccg ccatgcatta gttattaata gtaatcaatt
60 acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact
tacggtaaat 120 ggcccgcctg gctgaccgcc caacgacccc cgcccattga
cgtcaataat gacgtatgtt 180 cccatagtaa cgccaatagg gactttccat
tgacgtcaat gggtggagta tttacggtaa 240 actgcccact tggcagtaca
tcaagtgtat catatgccaa gtacgccccc tattgacgtc 300 aatgacggta
aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct 360
acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag
420 tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct
ccaccccatt 480 gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg
actttccaaa atgtcgtaac 540 aactccgccc cattgacgca aatgggcggt
aggcgtgtac ggtgggaggt ctatataagc 600 agagctggtt tagtgaaccg
tcagatggta ccatgtgtca ccagcagttg gtcatctctt 660 ggttttccct
ggtttttctg gcatctcccc tcgtggccat atgggaactg aagaaagatg 720
tttatgtcgt agaattggat tggtatccgg atgcccctgg agaaatggtg gtcctcacct
780 gtgacacccc tgaagaagat ggtatcacct ggaccttgga ccagagcagt
gaggtcttag 840 gctctggcaa aaccctgacc atccaagtca aagagtttgg
agatgctggc cagtacacct 900 gtcacaaagg aggcgaggtt ctaagccatt
cgctcctgct gcttcacaaa aaggaagatg 960 gaatttggtc cactgatatt
ttaaaggacc agaaagaacc caaaaataag acctttctaa 1020 gatgcgaggc
caagaattat tctggacgtt tcacctgctg gtggctgacg acaatcagta 1080
ctgatttgac attcagtgtc aaaagcagca gaggctcttc tgacccccaa ggggtgacgt
1140 gcggagctgc tacactctct gcagagagag tcagagggga caacaaggag
tatgagtact 1200 cagtggagtg ccaggaggac agtgcctgcc cagctgctga
ggagagtctg cccattgagg 1260 tcatggtgga tgccgttcac aagctcaagt
atgaaaacta caccagcagc ttcttcatca 1320 gggacatcat caaacctgac
ccacccaaca acttgcagct gaagccatta aagaattctc 1380 ggcaggtgga
ggtcagctgg gagtaccctg acacctggag tactccacat tcctacttct 1440
ccctgacatt ctgcgttcag gtccagggca agagcaagag agaaaagaaa gatagagtct
1500 tcaccgacaa gacctcagcc acggtcatct gccgcaaaaa tgccagcatt
agcgtgcggg 1560 cccaggaccg ctactatagc tcatcttgga gcgaatgggc
atctgtgccc tgcagttagg 1620 agctcataat cagccatacc acatttgtag
aggttttact tgctttaaaa aacctcccac 1680 acctccccct gaacctgaaa
cataaaatga atgcaattgt tgttgttaac ttgtttattg 1740 cagcttataa
tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt 1800
tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttaa cgcgaattcg
1860 gccggccgtt 1870 5 1417 DNA Artificial Sequence gene
(1)..(1417) Interleukin-7 (IL-7); Oligo DNA Dumbbell 5 ttcggccggc
caagcttaac cgtattaccg ccatgcatta gttattaata gtaatcaatt 60
acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat
120 ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat
gacgtatgtt 180 cccatagtaa cgccaatagg gactttccat tgacgtcaat
gggtggagta tttacggtaa 240 actgcccact tggcagtaca tcaagtgtat
catatgccaa gtacgccccc tattgacgtc 300 aatgacggta aatggcccgc
ctggcattat gcccagtaca tgaccttatg ggactttcct 360 acttggcagt
acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag 420
tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt
480 gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa
atgtcgtaac 540 aactccgccc cattgacgca aatgggcggt aggcgtgtac
ggtgggaggt ctatataagc 600 agagctggtt tagtgaaccg tcagatggta
ccatgttcca tgtttctttt aggtatatct 660 ttggacttcc tcccctgatc
cttgttctgt tgccagtagc atcatctgat tgtgatattg 720 aaggtaaaga
tggcaaacaa tatgagagtg ttctaatggt cagcatcgat caattattgg 780
acagcatgaa agaaattggt agcaattgcc tgaataatga atttaacttt tttaaaagac
840 atatctgtga tgctaataag gaaggtatgt ttttattccg tgctgctcgc
aagttgaggc 900 aatttcttaa aatgaatagc actggtgatt ttgatctcca
cttattaaaa gtttcagaag 960 gcacaacaat actgttgaac tgcactggcc
aggttaaagg aagaaaacca gctgccctgg 1020 gtgaagccca accaacaaag
agtttggaag aaaataaatc tttaaaggaa cagaaaaaac 1080 tgaatgactt
gtgtttccta aagagactat tacaagagat aaaaacttgt tggaataaaa 1140
ttttgatggg cactaaagaa cactgagagc tcataatcag ccataccaca tttgtagagg
1200 ttttacttgc tttaaaaaac ctcccacacc tccccctgaa cctgaaacat
aaaatgaatg 1260 caattgttgt tgttaacttg tttattgcag cttataatgg
ttacaaataa agcaatagca 1320 tcacaaattt cacaaataaa gcattttttt
cactgcattc tagttgtggt ttgtccaaac 1380 tcatcaatgt atcttaacgc
gaattcggcc ggccgtt 1417 6 28 DNA Artificial Sequence misc_feature
(1)..(28) DNA; single strandness; topology linear; "First
oligonucleotide, description; example 1.2" 6 aattcggccg gccgttttcg
gccggccg 28 7 28 DNA Artificial Sequence misc_feature (1)..(28)
DNA; single strandness; topology linear; "Second oligonucleotide,
description; example 1.2" 7 agcttggccg gccgttttcg gccggcca 28 8 27
DNA Artificial Sequence misc_feature (16)..(17) experimental DNA;
single strandness; topology linear; "First oligonucleotide,
description; example 3"; X=Amino- Uracil with via X coupled
peptide. 8 aattcggccg gccgtttcgg ccggccg 27 9 27 DNA Artificial
Sequence misc_feature (16)..(17) experimental DNA; single
strandness; topology linear; "Second oligonucleotide, description;
example 3"; X=Amino-Uracil with via X coupled peptide. 9 agcttggccg
gccgtttcgg ccggcca 27 10 27 DNA Artificial Sequence misc_feature
(16)..(17) experimental DNA; single strandness; topology linear;
"First oligonucleotide, description; example 4"; X=Thiol- Modifier
C6 S-S. 10 aattcggccg gccgtttcgg ccggccg 27 11 27 DNA Artificial
Sequence misc_feature (16)..(17) experimental DNA; single
strandness; topology linear; "Second oligonucleotide, description;
example 4"; X=Thiol-Modifier C6 S-S. 11 agcttggccg gccgtttcgg
ccggcca 27 12 27 DNA Artificial Sequence misc_feature (16)..(17)
experimental DNA; single strandness; topology linear; "First
oligonucleotide, description; example 6"; Y=Carboxy acid modified
thymidin desoxynucleotid. 12 aattcggccg gccgtttcgg ccggccg 27 13 27
DNA Artificial Sequence misc_feature (16)..(17) experimental DNA;
single strandness; topology linear; "Second oligonucleotide,
description; example 6"; Y=Carboxy acid modified thymidin
desoxynucleotid. 13 agcttggccg gccgtttcgg ccggcca 27
* * * * *